Abstract

Increased
longevity and improved health can be achieved in mammals by two feeding
regimens, caloric restriction (CR), which limits the amount of daily
calorie intake, and intermittent fasting (IF), which allows the food to be
availablead libitum every other day. The precise mechanisms
mediating these beneficial effects are still unresolved. Resetting the circadian clock is another
intervention that can lead to increased life span and well being, while
clock disruption is associated with aging and morbidity. Currently, a
large body of evidence links circadian rhythms with metabolism and feeding
regimens. In particular, CR, and possibly also IF,
can entrain the master clock
located in the suprachiasmatic nuclei (SCN) of the brain hypothalamus.
These findings raise the hypothesis that the
beneficial effects exerted by these feeding regimens could be mediated, at
least in part, through resetting of the circadian clock, thus
leading to synchrony in metabolism and physiology. This hypothesis is reinforced by a transgenic mouse
model showing spontaneously reduced eating alongside robust circadian
rhythms and increased life span. This review will summarize recent findings
concerning the relationships between feeding
regimens, circadian rhythms, and metabolism with implications for ageing
attenuation and life span extension.

I. Circadian rhythms,
well-being, and life span

Organisms on earth evolved to restrict their activity to the night or day, being
nocturnal or diurnal, respectively. By developing an endogenous circadian
(circa - about; dies - day) clock, which can be entrained to external stimuli, primarily light, animals and plants ensure that physiological processes are performed at the appropriate, optimal time of day or night [1]. Adaptation to external conditions through clock entrainment imparts a survival advantage, as the organism can predict environmental changes [1-3]. The clock core machinery is self sustained, so that in the absence of external cues, e.g., in constant darkness, the endogenous rhythms free-run, generating
cycles of approximately but not exactly 24 hours.

In mammals, the circadian clock influences nearly all aspects of physiology and behavior, such as sleep-wake cycles, cardiovascular activity, endocrine system, body temperature, renal activity, physiology of the gastrointestinal tract, and hepatic metabolism [1,2]. Epidemiological studies indicate that myocardial infarction, pulmonary edema, hypertensive crises, and asthma and allergic rhinitis attacks, all peak at certain times during the day [4-6]. Disruption of circadian coordination in humans or animals is manifested by hormone imbalance, some aspects of disease, and reduced life span [2,7-12].For instance, psychological and sleep disorders [2] and cardiovascular diseases [13,14] can be associated with irregular or dysfunctional circadian clock. Disruption of circadian coordination can also accelerate cancer proneness and malignant growth in animals and humans, suggesting that the circadian clock controls tumor progression [8-10]. In addition, symptoms seen in jet lagged travelers, e.g., fatigue, disorientation, and insomnia, or in shift workers, e.g., altered hormone profiles and morbidity, result from the constant need to extend wakefulness or to repeatedly invert the normal sleep-wake cycle [10,15,16]. Also, chronic reversal of the external light-dark cycle at weekly intervals resulted in a significant decrease in the survival time of cardiomyopathic hamsters [7]. Importantly, circadian rhythms change with normal aging in animals and humans, including a shift in the phase and decrease in amplitude [15-18]. By using a more direct approach, it was shown thatlongevity was diminished in golden hamsters carrying a 20 h-period mutation, tau, raised in 24 h light-dark cycles [19]. On the contrary, life span was extended in aged animals given fetal suprachiasmatic implants that restore higher amplitude rhythms [19-21]. Altogether, it seems that circadian disruption is associated with multiple negative manifestations, whereas resetting of circadian rhythms could lead to increased longevity. These findings, although largely correlative, point to a critical role for the circadian clock in maintaining normal peripheral physiology.

II. The circadian clock

A. The location of the mammalian circadian clock.
In mammals, the central circadian clock is located in the suprachiasmatic nuclei (SCN), a distinct bilateral group
of cells located in the anterior hypothalamus in the brain. Similar clock oscillators have been found
in many peripheral tissues, such as the liver, intestine, heart, adipose tissue, retina and in various
regions of the brain [2,22-24].
The SCN clock is composed of multiple, intracellular circadian oscillators, which, when synchronized,
generate coordinated circadian outputs that regulate overt rhythms [25-28].
SCN oscillation is not exactly 24 h and it is necessaryto entrain the circadian pacemaker each day to the external
light-dark cycle to prevent drifting (or free-running) out of phase. Light perceived primarily by melanopsin-expressing
retinal ganglion cells transmit signals to the SCN via the retinohypothalamic tract (RHT)
[2,29,30].
As a result, vasoactive intestinal polypeptide (VIP), an intrinsic SCN factor, acutely activates and synchronizes
SCN neurons [31,32]. Synchronization among SCN neurons leads
to the sending of signals to peripheral oscillators to prevent the dampening of circadian rhythms in these tissues.
The SCN accomplishes this task via neuronal connections or circulating humoral factors [33]
although the mechanisms are not fully understood (Figure 1). Several humoral factors expressed cyclically by the SCN,
such as transforming growth factor α (TGFα) [34], prokineticin 2 (PK2) [35],
and cardiotrophin-like cytokine (CLC) [36], have been shown to affect peripheral clocks.
Their intracerebroventricular injection inhibits nocturnal locomotor activity, an SCN output.
Complete electrical destruction of SCN neurons abolishes overall circadian rhythmicity in SCN-controlled tissues,
because of the loss of synchrony among individual cells in the periphery and damping of the rhythm at the population level
[37]. However, at the cellular level each cell oscillates, but with
a different phase [37,38].
The fraction of cyclically expressed transcripts in each peripheral tissue ranges between 5-20% of the total
population and the vast majority of these genes are tissue-specific [24,39-47].
These findings emphasize the circadian control over a large portion of the transcriptomes in peripheral tissues.
Considering the circadian gene expression in peripheral tissues, it is difficult to determine whether the SCN
clock drives these rhythmic patterns directly or indirectly by driving rhythmic feeding, activity, and/or body temperature,
which, in turn, contribute to rhythms in gene expression in the periphery.
It has been shown that for a peripheral tissue, such as the liver, signals both from the SCN clock or the local
endogenous clock may control rhythmic gene expression [48,49].

B. The biological clock at the
molecular level.
Genetic analysis of mutations
affecting the clock in Neurospora, Drosophila, Cyanobacteria, Arabidopsis,
and, recently, the mouse have paved the way for the identification of clock
genes. In mammals, the clock is an intracellular mechanism sharing the same
molecular components in SCN neurons and peripheral cells [3]. Generation of
circadian rhythms is dependent on the concerted co-expression of specific clock
genes. Transcriptional-translational feedback loops
lie at the very heart of the core clock mechanism. Many clock gene
products function as transcription factors, which
possess PAS (PER, ARNT, SIM) and basic helix-loop-helix (bHLH) domains
involved in protein-protein and protein-DNA interactions, respectively. These
factors ultimately activate or repress their own
expression and, thus, constitute self-sustained transcriptional feedback loops. Changes in concentration, subcellular localization, post-transcriptional microRNA
regulation, posttranslational modifications (phosphorylation, acetylation,
deacetylation, SUMOylation), and delays between transcription and
translation are crucial in order to achieve a 24-h cycle [1,2,50-52].

Figure 1. Resetting signals of the central and peripheral clocks.
The SCN resets peripheral oscillators via
humoral factors or autonomic innervation leading to circadian hormone
expression and secretion and rhythmic activity of metabolic pathways. In
addition, the SCN dictates rhythms of locomotor activity, sleep-wake cycle,
blood pressure, and body temperature. Light, food, and feeding regimens
affect either the central clock in the SCN or peripheral clocks. Input to
central or peripheral clocks are in blue. Outputs from the central clock to
the periphery are in green.

In the mouse, the first clock gene identified, encodes the
transcription factor CLOCK (Circadian Locomotor
Output Cycles Kaput) [53], which dimerizes with BMAL1 (brain and muscle ARNT-like protein 1) to activate
transcription. CLOCK and BMAL1, two bHLH-PAS
transcription factors, are capable of activating transcription upon binding to
E-box (5'- CACGTG -3') and E-box-like promoter sequences [2]. BMAL1 can also
dimerize with other CLOCK homologs, such as neuronal PAS domain protein
2 (NPAS2), to activate transcription and sustain
rhythmicity [54,55]. Amongst the regulatory targets of CLOCK:BMAL1 are
the three Period genes (Per1, Per2, and Per3),
which encode PAS domain factors, and two Cryptochrome genes (Cry1
and Cry2). PERs and CRYs function as negative regulators, blocking
CLOCK:BMAL1-mediated transcriptional activation [2,56] (Figure 2A).Thus, CLOCK:BMAL1
heterodimers bind to E-box sequences and mediate transcription of a large number of genes including those of
the negative feedback loop Pers and Crys. When PERs and CRYs are
produced in the cytoplasm, they
oligomerize after reaching an appropriate concentrationand translocate to the nucleus to inhibit
CLOCK:BMAL1-mediated transcription. All the aforementioned clock genes
exhibit a 24-h oscillation in SCN cells and peripheral tissues, except for Clock
that has been shown not to oscillate in the SCN [50]. Recent studies have
demonstrated that CLOCK has intrinsic histone acetyltransferase activity,
suggesting that rhythmic activation of chromatin remodeling may underlie the
clock transcriptional network [57,58]. Indeed, cyclic histone acetylation and
methylation have been observed on the promoters of several clock genes [58-63].
In addition, CLOCK also acetylates several proteins of the core clock
apparatus, thus, enabling cycles of acetylation and deacetylation, the latter
activity involving SIRT1 will be discussed below (Figure 2).

Figure 2. The core mechanism of the mammalian circadian clock and its link to energy metabolism.
(A) High NAD(P)H levels promote
CLOCK:BMAL1 binding to E-box sequences leading to the acetylation of BMAL1
and expression of Pers, Crys, and other clock-controlled genes. The
negative feedback loop, PERs:CRYs, binds to CLOCK:BMAL1 and consequently
PERs are acetylated. Activated AMPK leads to a rise in NAD+ levels,
phosphorylation of CRYs, and phosphorylation of CKI?, which then phosphorylates
the PERs. As a result of increased NAD+ levels, SIRT1 deacetylates PERs and BMAL1.
This and the destabilization of phosphorylated PERs and CRYs relieves PERs:CRYs
repression and another cycle starts. (B) Expression of Bmal1 and Rev-erbα
genes are controlled by PPARα and binding of RORs to RORE sequences. RORs
need a co-activator, PGC-1α, which is phosphorylated by activated AMPK. In
parallel, AMPK activation leads to an increase in NAD+ levels, which, in turn
activate SIRT1. SIRT1 activation leads to PGC-1α deacetylation and activation.
Acetyl adenosine diphosphate ribose (Ac-ADP-r) and nicotinamide (NAM) are released
after deacetylation by SIRT1.

Several other players appear
to be important to sustain clock function. Casein kinase I
epsilon (CKIε) phosphorylates the PER proteins and, thereby, enhances
their instability and degradation [50,64-66]. CKIε also phosphorylates and
partially activates the transcription factor BMAL1 [67]. Bmal1
expression is nega?tively regulated by the transcription factor reverse
erythroblastosis virus α (REV-ERBα)
[68], which recruits histone
deacetylase (HDAC) complexes [69]. Bmal1 expression is positively regulated by retinoic acid receptor-related orphan
receptor α (RORα) and RORγ [70]via the ROR response element (RORE) [71]. Thus, Bmal1 oscillation is driven by a rhythmic change in
RORE occupancy by RORs and REV-ERBα. This alternating occupancy occurs due
to the robust rhythmic levels of REV-ERBα, a result of direct
transcriptional activation of the Rev-erbα gene by the heterodimer
CLOCK:BMAL1 [68]. Indeed, mice
deficient in RORα or REV-ERBα have impaired circadian rhythms of
locomotor activity and clock gene expression [68,70] (Figure 2B).

III. The biological clock and energy homeostasis

A. Circadian rhythms and metabolism

The circadian clock has been reported to regulate
metabolism and energy homeostasis in peripheral tissues [72,73]. This is
achieved by mediating the expression and/or activity of certain metabolic
enzymes and transport systems [74,75] involved in metabolic pathways [76-80].
In addition, a large number of nuclear receptors involved in lipid and glucose
metabolism has been found to exhibit circadian expression [81]. Many hormones
involved in metabolism, such as insulin [76], glucagon [82], adiponectin [83],
corticosterone [84], leptin, and ghrelin [85,86], have been shown to exhibit
circadian oscillation. Leptin, an adipocyte-derived circulating hormone, acts
at specific receptors in the hypothalamus to suppress appetite and increase
catabolism. Leptin exhibits striking circadian patterns in both gene expression
and protein secretion, with peaks during the sleep phase in humans [87].
Neither feeding time nor adrenalectomy affected the rhythmicity of leptin
release. However, ablation of the SCN has been shown to eliminate leptin
circadian rhythmicity in rodents, suggesting that the central circadian clock
regulates leptin expression [88]. Receptors for leptin and ghrelin are present
on SCN cells [89-91], so it is possible that these hormones bind directly to
SCN neurons, similarly to their effect on the orexigenic neuropeptide Y (NPY)
and agouti-related protein (AgRP) neurons. Indeed, exogenous leptin was
reported to phase-advance rhythms of neuronal firing in rat SCN slices [92].Activation of ventromedial arcuate nucleus (vmARC) neurons by systemic
administration of the ghrelin mimetic growth hormone-releasing peptide 6 combined
with SCN tracing showed that vmARC neurons transmit feeding-related signals to
the SCN [90]. This injection induced Fos in the vmARC and resulted in
attenuation of light-induced phase delay in mice and light-induced Fos
expression in the SCN in rats [93]. Administration of ghrelin in vitro
to SCN slices or SCN explants from Per2::luc transgenic mice caused
phase shifts in Per2::luc reporter gene expression. However, intra-peritoneal
injection of ghrelin to wild type mice caused phase shifts only after 30 h of
food deprivation, but not when the mice were fed ad libitum [94]. Thus,
it seems that ghrelin and leptin may affect the SCN directly or through their
effect on the ARC, which is then relayed to the SCN.

Experiments using cultured cells have suggested that the cellular redox state is
capable of influencing rhythms [95]. CLOCKand itshomolog
NPAS2 can bind efficiently to BMAL1 andconsequently to E-box sequences in the
presence of reduced nicotinamide adenine dinucleotides (NADH and NADPH)
(Figure 2A). On the other hand, the oxidized forms of the nicotinamide adenine
dinucleotides (NAD+ and NADP+) inhibit DNA binding of
CLOCK:BMAL1 or NPAS2:BMAL1 [95,96].
As the NAD(P)+/NAD(P)Hredox equilibrium depends on the metabolicstate of
the cell, this ratio could dictate the binding of CLOCK/NPAS2:BMAL1 to E-boxes and
result in phase-shifting of cyclic gene expression [74,95,96].
NAD+ is also required for the activation of SIRT1, a deacetylase involved in
clock modulation, as will be discussed below.

BMAL1: Circadian clocks have been shown to be present in several fat tissues, including inguinal white adipose tissue, epididymal white adipose tissue, and brown adipose tissue [45,97,98]. Recent transcriptome studies revealed rhythmic expression of clock and adipokine genes, such as resistin, adiponectin, and visfatin, in visceral fat tissue [83]. Recent molecular studies established the involvement of BMAL1 activity in the control of adipogenesis and lipid metabolism in mature adipocytes. Embryonic fibroblasts from Bmal1-/- knockout mice failed to differentiate into adipocytes. Loss of BMAL1 expression led to a significant decrease in adipogenesis and gene expression of several key adipogenic/lipogenic factors. Furthermore, over-expression of BMAL1 in adipocytes increased lipid synthesis activity. Thus, BMAL1, a master regulator of circadian rhythms, plays important roles in the regulation of adipose differentiation and lipogenesis in mature adipocytes [99].

REV-ERBα: Another important candidate to link the circadian clock with lipid metabolism is REV-ERBα. This pro-adipogenic transcription factor, whose levels increase dramatically during adipocyte differentiation [100], exhibits striking diurnal variations in expression in murine adipose tissue [101] and rat liver [102]. During adipocyte differentiation, REV-ERBα acts downstream of the differentiation factor peroxisome proliferator receptor-γ (PPARγ) by facilitating gene expression of PPARγ target genes [103,104]. Ectopic REV-ERBα expression in 3T3L1 pre-adipocytes promotes their differentiation into mature adipocytes [103]. In addition to its role in lipid metabolism and adipocyte differentiation, REV-ERBα is a component of the core clock apparatus, as mentioned above (Figure 2B). It acts as a negative regulator of Bmal1 expression, and its encoding gene, Rev-erbα, is directly activated by the CLOCK:BMAL1 heterodimer [68].

PPARα: Peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear receptor family that plays a unique role at the intersection of circadian and lipid metabolic pathways. The CLOCK:BMAL heterodimer mediates the expression of PPARα, which subsequently binds to the peroxisome-proliferator response element (PPRE) and activates transcription of Bmal1 [105-107] (Figure 2B). PPARα also regulates transcription of genes involved in lipid and glucose metabolism upon binding of endogenous free fatty acids [108,109]. Thus the circadian rhythmicity of PPARα provides an example of a reciprocal link between circadian and lipid metabolic processes.

PPARγ coactivator (PGC-1α): PGC-1α, a transcriptional co-activator that regulates energy metabolism, is rhythmically expressed in the liver and skeletal muscle of mice. PGC-1α stimulates the expression of Bmal1 and Rev-erbα, through co-activation of the ROR family of orphan nuclear receptors [110,111] (Figure 2B). Mice lacking PGC-1α show abnormal diurnal rhythms of activity, body temperature, and metabolic rate, due to aberrant expression of clock genes and those involved in energy metabolism. Analyses of PGC-1α-deficient fibroblasts and mice with liver-specific knockdown of PGC-1α indicate that it is required for cell-autonomous clock function [110].

AMP-activated protein kinase (AMPK): AMPK could be another important link that integrates the circadian clock with metabolism. AMPK is a sensor of the energy status within cells, which upon activation acts to restore energy balance [112,113]. This is done in part by modulating NAD+ levels and SIRT1 activity [114,115]. AMPK has been found to directly phosphorylate Ser-389 of CKIε in Rat-1 fibroblasts, resulting in increased CKIε activity and degradation of mPER2 (Figure 2A). mPER2 degradation led to a phase advance in the circadian expression pattern of clock genes [116]. AMPK has also been shown to phosphorylate and destabilize mCRY1 in mouse fibroblasts, leading to altered circadian rhythms [117] (Figure 2A). In addition, the expression profile of clock-related genes, such as Per1 and Cry2 in skeletal muscle, as well as the diurnal shift in energy utilization, is impaired in AMPKγ3 subunit knockout mice in response to 5-amino-4-imidazole-carboxamide riboside (AICAR), an AMPK activator [118]. In addition to its intracellular role, AMPK is involved in whole body energy metabolism by regulating the response to feeding in the hypothalamus [112]. In this brain area, AMPK activation is inhibited by leptin and insulin, hormones which suppress feeding, whereas it is activated under starvation by ghrelin, a hormone primarily produced by the stomach that leads to increased food intake [119-122].

SIRT1: Another protein recently found to link metabolism
with the circadian clock is SIRT1. This is the mammalian ortholog of yeast
Sir2, an NAD+-dependent histone deacetylase involved in
transcriptional silencing and genome stability in yeast [123,124]. Sir2 or its
ortholog enzymes are involved in life span extension and the response to
caloric restriction in yeast, Caenorhabditis
elegans, Drosophila [123,125], and mice [115,126]. The dependence on NAD+ as a cofactor for catalysis is thought
to link SIRT1 activity to the energy state of the cell [127]. Non-histone
substrates of SIRT1, as found in C2C12 myotubes, include regulatory molecules
that modulate energy metabolism, such as PPARγ and PGC-1α [114], key
factors that regulate the core molecular clock (Figure 2). Recent studies showthat SIRT1 interacts directly with CLOCK and
deacetylates BMAL1 and PER2 in cultured fibroblasts [128,129] (Figure 2A). It
seems that after binding to E-box, CLOCK and CBP/p300 acetylate histones H3 and
H4 [57] and BMAL1 [130]. BMAL1 acetylation potentiates its binding by the
repressive PER/CRY complex [130] and, as a result, PER2 is acetylated [128].
When acetylated, PER2 [128] and possibly BMAL1 [129] are more stable. SIRT1
then becomes activated and deacetylates BMAL1, PER2, and histones [131].
Deacetylated PER2 is further phosphorylated and degraded and a new cycle begins
(Figure 2A). It has also been shown that the CLOCK:BMAL1 heterodimer regulates
the circadian expression of NAMPT (nicotinamide phosphoribosyl-transferase), a
rate-limiting enzyme in the NAD+ salvage pathway. SIRT1 is recruited
to the Nampt promoter and contributes to the circadian synthesis of its
own coenzyme [132]. Most recently, it has been shown that AMPK enhances SIRT1
activity by increasing cellular NAD+ levels, resulting in the
deacetylation and modulation of the activity of downstream SIRT1 targets [114].
Thus, the levels of NAD+ together with the cycling of SIRT1 can
determine the activity and robustness of clock gene transcription at least in
cultured cells.

C. Clock mutations and metabolic disorders

The most compelling linkage between metabolic
disorders and the circadian clock is demonstrated by the phenotypes of clock
gene mutants and knockouts. Homozygous C57BL/6J ClockΔ19 mice,
with a truncated exon 18 and deleted exon 19 of the Clock gene, have a
greatly attenuated diurnal feeding rhythm, are hyperphagicand
obese, and develop a metabolic syndrome of hyperleptinemia,hyperlipidemia,
hepatic steatosis, and hyperglycemia [133]. Loss of circadian rhythms in ClockΔ19 mutant
mice was accompanied by attenuated expression of hypothalamic peptides associated
with energy balance, such as ghrelin and orexin [133]. Insulin administration
caused significantlygreater hypoglycemia in ClockΔ19 mutant
mice than in wildtype mice [134]. Increased insulin sensitivity was
also seen in ClockΔ19 mutant mice of the BALB/c/CBA
background that preserve rhythmicity in melatonin production [135]. In ClockΔ19 on an
Jcl:ICR background, serum levels of triglyceride and free fatty acids were
significantly lower than in wild type control mice, whereas total cholesterol
and glucose, insulin, and leptin levels did not differ [136]. Unlike C57BL/6J
ClockΔ19 mutant
mice, Jcl:ICR ClockΔ19 mutantmice were not obese, had low or
normal fasting plasmaglucose, low plasma free fatty acids, and
normal plasma leptin. However, in Jcl:ICR
ClockΔ19 mutant mice, high
fat diet amplified the diurnal variation in glucose tolerance and insulin
sensitivity, and obesity was attenuated through impaired dietary fat absorption
[136]. Although the effects on metabolism were variable due to strain
differences, the overall picture is that disruption of the clock gene leads to
disruption of metabolic pathways.

Bmal1-/- knockout mice, similarly to C57BL/6J ClockΔ19mutant
mice, exhibited suppressed diurnal variations in glucose and triglycerides as
well as abolished gluconeogenesis. Liver-specific deletion of Bmal1
showed a direct effect of the liver clock on glucose metabolism, as exhibited
by hypoglycemia during fasting, exaggerated glucose clearance, and loss of
rhythmic expression of hepatic glucose regulatory genes [137]. Although
recovery from insulin-induced hypoglycemia was impaired in C57BL/6J ClockΔ19 mutant
and Bmal1-/- knockout mice, the counter-regulatory responses
of corticosterone and glucagon were retained [134].

Mutation in another central clock gene, Per2 (mPer2-/-
mice), exhibited no glucocorticoid rhythm even though the corticosterone
response to hypoglycemia was intact. In addition, the diurnal feeding rhythm
was absent in these mice. Although food consumption was similar during the
light and dark periods on high fat diet, mPer2-/- mice
developed significant obesity [138].

IV. Effect of feeding regimens on circadian rhythms

In addition to light, feeding regimens have been reported to affect the clocks in the SCN and/or the periphery.

A. Restricted feeding (RF)

RF limits the time and duration of food availability with no calorie reduction [3,74,139].Animals, which receive food ad libitum everyday at the same time for only a few hours, adjust to the feeding period within a few days [49] and can consume their daily food intake during that limited time [140,141]. Restricting food to a particular time of day has profound effects on the behavior and physiology of animals. Many physiological activities that are normally dictated by the master clock in the SCN are altered by RF, such as hepatic P450 activity, body temperature, locomotor activity, and heart rate [142-145]. 2-4 h before the meal, the animals display food anticipatory activity (FAA), which is typifiedby an increase in locomotor activity, body temperature, corticosterone secretion,gastrointestinal motility, and activity of digestive enzymes [140,146-148], all are known output systems of the biological clock. RF is dominant over the SCN and drives rhythms in arrhythmic and clock mutant mice and animals with lesioned SCN, regardless of the lighting conditions [142,143,148-151]. In most incidents, RF affects the core clock apparatus in peripheral tissues, such as liver (Figure 3), kidney, heart, and pancreas, with no effect on the central pacemaker in the SCN [3,74,139,143,150,152,153], causing uncoupling from the central pacemaker in the SCN. This suggests that nutritional regulation of clock oscillators in peripheral tissues may play a direct role in coordinating metabolic oscillations [154]. As soon as food availability returns to normal, the SCN clock, whosephase remains unaffected,resets the peripheral oscillators [152]. The location of this food-entrainable oscillator (FEO) has been elusive. Lesions in the dorsomedial hypothalamic nucleus (DMH) [155-158], the brain stem parabrachial nuclei (PBN) [156,159], and the core and shell regions of nucleus accumbens [160,161] revealed that these brain regions may be involved in FEO output, but they cannot fully account for the oscillation [162]. Neither vagal signals nor leptin are critical for the entrainment [163,164].CLOCK [165] or BMAL1 [166] and other clock genes [167] have been shown not to be necessary for food anticipatory activity. However, it has recently been demonstrated that mPer2 mutant mice did not exhibit wheel-running food anticipation [168,169]. Recently, the FEO was suggested to be localized, in part, in ghrelin-secreting cells in the stomach [170]. Clearly, the localization and nature of the FEO and the effect of RF on circadian rhythms warrants further study.

The effect of RF on ageing and longevity has never been studied. Interestingly, the survival of Glasgow osteosarcoma-inoculated mice was prolonged under an RF regimen during the light period compared to those under the dark period or those fed ad libitum [171]. Also, RF modified the expression of genes involved in carcinogenesis and tumor progression, such as c-myc and p53 [172]. It remains to be determined whether RF feeding affects life span.

B. Calorie restriction (CR)

CR, sometimes denoted dietary restriction (DR), refers
to a dietary regimen low in calories without malnutrition, that restricts the
daily amount of calories derived from carbohydrates, fats, or proteins
usually to 60-75% of ad libitum-fed animals.
CR extends the life span of diverse species, such as C. elegans, Drososphila,
rodents [125,173,174], and recently monkeys [175]. CR in mice, rats,
and monkeys prevents or delays the onset of major age-related diseases, such as
cancer, diabetes, kidney disease, and cataracts [173,176]. In humans,
long-termed CR results in sustained beneficial effects on major risk factors
for atherosclerosis, type 2 diabetes, and inflammation [177]. The reduction of
energy intake is considered to be the critical beneficial factor in the CR
regimen [173]. Theories on how CR modulates aging and longevity abound, but the
exact mechanism is still unclear [178]. For a longtime,
the most prevalent explanation was related to the widely acceptable theory on
aging, the Free Radical Theory. This theory attributes the aging process to the
continuousaccumulation of oxidative damage
in macromolecules generated by reactive oxygen species (ROS) produced in the
mitochondria [179]. A later variation of this theory, the Oxidative
Stress Theory, attributes the oxidative damage to the imbalance between
preoxidant and antioxidant components, and CR was suggested to increase the
resistance to oxidative stress [180]. Recently, this explanation was put into
question, at least for rodents, as increasing oxidative stress by several
genetic alterations increased aging-related diseases, such as cancer, but did
not diminish life span [181-183]. ROS, primarily H2O2,
have recently been suggested to promote aging as activators of the TOR (target
of rapamycin) pathway [184]. This
signaling pathway acts as a sensor of the nutritional and energetic state in
the cell and transmits anabolic signals to regulate cell size, growth, and
metabolism. Mammalian TOR (mTOR) could play an important role in the regulation
of life span, as indicated by findings showing that CR attenuated mTOR
signaling in several tissues in mice [185], and mice
deficient of ribosomal protein S61 kinase 1, a central component in mTOR signaling, or mice treated with rapamycin, an inhibitor of the mTORC1 component, exhibited
increased life span [186,187].
Interestingly, mTOR has also been recently linked to the circadian clock as a
light-activated signaling cascade in the SCN of mice [188].

Figure
3. Effect
of night vs. day RF and night vs. day IF on clock gene expression. Expression
of a representative clock gene mCry1 was measured in the liver of
C57BL mice during
ad libitum (AL), day and night RF, and day and night IF.Total
RNA extracted from liver tissue collected every 3 h around the circadian
cycle (mean ± SEM; n=3 for each time-point and each mouse group) was
reverse transcribed and analyzed by quantitative real time PCR. Clock gene levels
were normalized using Gapdh as the reference gene. The grey and
black bars designate the subjective light and dark cycles, respectively.

CR-fed animals resemble RF-treated animals, as they
usually consume all or most of their food within a short period of time. While
anticipating for food, calorically restricted animals show a rise in body
temperature [189]. Thus, due
to the temporal component of food intake, it is possible that CR, similarly to
RF, synchronizes peripheral clocks and influences clock-controlled output
systems, such as the anticipatory behavior and body temperature. As opposed to
RF, CR entrains the clock in the SCN [190-192]. Under
light-dark conditions and daytime feeding, calorically restricted mice showed
strong FAA but with a phase advance of the nocturnal pattern of activity, a
direct output of the SCN clock. When mice were transferred to dark-dark
conditions, i.e. under free-running conditions, and fed ad libitum, the
onset of the nocturnal period of locomotor activity occurred significantly
earlier (1.3 h) in the calorically restricted than in ad libitum-fed
animals, indicating an SCN effect. The period, however, did not differ between
calorically restricted and ad libitum-fed mice [190]. Also,
when SCN clock gene expression was tested, slight changes in gene expression
were observed [191,192]. Overall, these results suggest that CR during the
daytime affects the temporal organization of the SCN clockwork and circadian outputs in mice under light-dark cycle. In
addition, CR affects photic responses of the circadian system, as measured by
light pulses, suggesting that energy metabolism modulates gating of photic
inputs in mammals [192]. It is noteworthy that
microarray data comparing gene expression in seven different tissues under CR
identified circadian rhythms among the three most over-expressed biological
processes, with Per2 being the most up-regulated gene [193]. Collectively,
these findings suggest that synchronization of peripheral oscillators
during CR could be achieved directly due to the temporal feeding, as has been
reported for RF [143,152,153], or by synchronizing the SCN [190-192], which, in turn, sends humoral
or neuronal signals to entrain the peripheral tissues [194,195]. It is not known whether there
is dominancy or harmony between the central pacemaker and peripheral
oscillators under CR.

C. Intermittent fasting (IF)

During
IF, also denoted alternate day fasting (ADF), food is available ad libitum
every other day. IF-treated mice eat on the day they have access to food
approximately twice as much as those having continuous access to food [196-198]. Similarly to
calorically restricted animals, IF-fed animals exhibit increased life span in
comparison with the ad libitum-fed control, even if there is little or
no overall decrease in calories [199,200]. IF-fed
animals also exhibit improved glucose metabolism, cardio-protection,
neuro-protection [196,201-205], and increased
resistance to cancer [197,200]. IF may also
decrease the risk for cardiovascular diseases in humans [206].The IF-induced beneficial effects are thought to
occur independently of the overall caloric intake, but the underlying
mechanisms are still unknown. One suggested mechanism is stimulation of
cellular stress pathways induced by the IF regimen [196,207,208]. Brain-derived
neurotrophic factor (BDNF), normally involved in brain development and
plasticity, is elevated in IF animals, and is causally linked to the protective
effect of the IF regimen against neuronal damage inflicted by the neurotoxin
kainic acid [209]. It must be
noted, however, that BDNF could not be linked to the neuro-protective effects
in the brain of calorically restricted rats [210,211], but increased
levels of another neurotrophic factor, glial cell line-derived factor (GDNF),
were correlated with neuro-protection of a calorically restricted primate model
of Parkinson's disease [212]. Interestingly,
BDNF is also a component of the hypothalamic melanocortin pathway that controls
food intake and body weight in adult mice [213], and it
has been implicated in the regulation of energy metabolism [214]. Heterozygousknockout BDNF (BDNF+/-) mice exhibit metabolic abnormalities,
hyperphagia, obesity, and insulin resistance that could be significantly
reversed by IF, indicating that BDNF is indeed involved in the beneficial
effects induced by IF [214]. Interestingly,the BDNF+/- mice resemble circadian Clock mutant
mice [133] in metabolic
abnormalities. In addition, BDNF and its cognate receptor TrkB were suggested
to play a role in circadian modulation of the SCN pacemaker sensitivity to
light [215,216]. These
data point to the possibility that IF could affect the SCN and, as a result,
peripheral clocks, at least via elevating BDNF levels.

Recently, we have shown that, under an IF protocol,when food was introduced during the light period,
mice exhibited almost arrhythmicity in clock gene expression in the liver.
Unlike daytime feeding, nighttime feeding yielded rhythms similar to those
generated during ad libitum feeding [198] (Figure 3). Furthermore, rhythms were maintained when
daytime IF occurred under disruptive light, suggesting that SCN signals were
involved in inducing the arrhythmic state in the periphery [198]. Thus, the fact that IF can affect circadian rhythms
differently depending on the timing of food availability and light conditions
suggests that this regimen affects the SCN clock, similarly to CR. We assume
that SCN resetting by IF and CR could be involved in the health benefits
conferred by these regimens [195].

The effects of
IF are in contrast to those of restricted feeding (RF) that dictates peripheral
rhythms in arrhythmic and mutant mice and animals with lesioned SCN regardless
of the lighting conditions [142,143,148-151]
(Figure 3). It, thus, appears that IF is not as dominant as RF in dictating
peripheral rhythms. Never-theless, this feeding regimen exhibits some
similarities with RF, as reflected by the anticipatory feeding behavior that
preceded food availability and restoration of circadian rhythms under
disruptive light conditions, due most likely to the effect on the food
entrainable oscillator (FEO) [198]. Thus, under
daytime IF, clock gene expression in the periphery would be controlled by the
SCN, which responds to both light-dark cycle and IF, as well as directly by the
temporal feeding via the FEO. Co-activation of both the FEO and the SCN
would yield rhythms at two opposite phases leading to overall arrhythmicity. In
contrast, under nighttime IF, normal rhythms are generated, as both the FEO and
the SCN work in synchrony to dictate peripheral rhythms [198].

V. The circadian
clock as a possible mediator in CR- or IF-induced increased longevity

A.
Long-lived, spontaneously calorically restricted αMUPA mice

αMUPA mice carry as a transgene, the urokinase-type
plasminogen activator (uPA) [217], an extracellular fibrinolytic serine protease
implicated in tissue remodelling [218] and brain
development and plasticity [219-224]. αMUPA mice
spontaneously eat less (20-30%) compared to their wild type (WT) FVB/N control
mice when fed ad libitum, indicating that their appetite is genetically
suppressed. The mechanism linking transgenic uPA to reduced hunger is not yet
clear. It could be related to uPA over-expression in the brain stem, as was
found in two transgenic lines showing reduced food intake [225]. The transgenic
effect is likely to be developmental, similarly to the remodelling effect
recently detected in αMUPA developing incisor teeth [226]. αMUPA
mice live longer (median, 16%; 10th decile, 15%) than WT mice [227], thus resembling
calorically restricted animals in showing an inverse relation betweenfood intake and life span.αMUPA
mice exhibit additional similarities with calorically restricted mice, such as
reduced body weight, reduced levels of serum IGF-1 or glucose, enhanced
capacity to conduct apoptosis, and reduced incidence of tumors[225,227-230].

B.
αMUPA mice, circadian rhythms, and aging

Recent
data show that αMUPA mice exhibit higher amplitude in the circadian
expression of several clock genes in the liver compared with FVB/N WT mice.
This change
coincides with higher amplitude rhythms of food intake and body temperature [194].
Since circadian patterns of food intake and body temperature constituteclock-controlled output systems, it is
conceivable that their alteration in the transgenic
mice stems from the higher amplitude of clock gene expression in the periphery,
and possibly also in the central biological clock in the SCN. Higher amplitude
of circadian rhythms have been previously associated with young age [15] and
extended life span [19].
Support for a linkage between circadian rhythms and attenuation of aging in
αMUPA mice is provided by comparing young vs. old mice. When tested for
circadian food intake, an SCN output system, 18-month-old WT control mice
exhibit a 4-6-h shift in circadian food intake compared to 5-month-old mice [195].
This behaviour is consistent with literature data showing that aging can alter
the amplitude and/or phase of circadian rhythms [15,16,18].
In contrast to WT mice, adult and young αMUPA mice show similar circadian
food intake, indicating that at least some aspects of circadian behavior
maintain a youthful pattern at an old age in these mice. At an old age,
αMUPA mice maintain a young and healthier
appearance, they look lean, and their fur is shiny, whereas WT mice are
sluggish and they look old (Figure 4). In addition, αMUPA
mice do not become obese throughout their life-time, whereas about one third of
the WT mice show severe obesity (Figure 4). The major difference in body weight
between αMUPA and WT mice stems from the fact that the quantitative
difference in food intake between αMUPA and WT mice is maintained at the
old age.

C.
αMUPA mice reveal effectsof feeding regimens
on circadian rhythms

It is difficult
to eliminate the effect of temporal food consumption in calorically restricted
animals, as mice consume their food within a few hours. αMUPA mice spontaneously
consume reduced calories (20-30% reduction) compared with WT mice under
different feeding regimens, i.e. AL, RF and IF, suggesting that these mice can
be utilized as a model for CR in the absence of the imposed temporal food
consumption under ad libitum feeding, and a model for imposed temporal CR under
RF or IF conditions. Therefore,
the transgenic mouse model αMUPA [225] has recently
been used to investigate the contribution of calorie reduction per se vs.
timed feeding to clock adaptation [49]. Under light-dark
conditions and ad libitum feeding, αMUPA mice show high amplitude,
appropriately reset circadian rhythms in peripheral clock genes [194] (Figure 5). This finding
could reflect the effect of the reduced calorie intake on the SCN in αMUPA
mice, as has been previously reported for calorically restricted animals [190-192].

Figure
5.
Clock gene expression in the liver under various feeding and lighting
conditions in αMUPA (M) and WT mice. Expression levels
of the following clock genes are presented under light-dark or disruptive
light conditions:mPer2during ad
libitum (AL) feeding, mCry1 under RF, and Bmal1 under IF.Total
RNA extracted from liver tissue collected every 3 h around the circadian
cycle (mean ± SEM; n=3 for each time-point and each mouse group) was
reverse transcribed and analyzed by quantitative real time PCR. Clock gene levels
were normalized using Gapdh as the reference gene.

Under light-dark
conditions, RF advanced the expression phase of all clock genes in the liver in
a similar manner in both WT mice and αMUPA mice, and in some cases
increased the amplitude [49](Figure 5). These findings
were in concert with previous results in the literature, as is mentioned
above. Nevertheless, an effect of the SCN on clock-controlled output systems
under RF could be seen when the phases of peripheral clock gene expression in
WT mice vs. αMUPA mice were compared under arrhythmicity imposed by
disruptive light. Whereas the pattern of clock gene expression did not change
in WT mice, a phase shift was seen in several genes in αMUPA mice (Figure
5), suggesting an SCN effect. Again, this effect of the SCN could be
attributed to the reduced calorie intake of αMUPA mice. Altogether,
these findings suggest that both the reduced calories and the light-dark cycle
work in synchrony on the central biological clock of αMUPA mice to
generate rhythms in the periphery. However, it
seems that in WT mice, as has been found for other mouse strains, RF is
dominant over the SCN in dictating rhythms in the periphery regardless of the
lighting conditions. In αMUPA mice, RF dictates the phase of clock gene
expression under light-dark; but under disruptive light conditions, as the SCN
is under the influence of calorie restriction, it becomes dominant in dictating
the phase of clock gene expression.

Unlike RF, the
effect of IF on circadian rhythms in αMUPA mice was similar to that of WT
mice under light-dark or disruptive light conditions, and it resembled also
that on C57BL mice (Figure
3, Figure 5).
Thus, in all mice tested, daytime IF caused arrhythmia in clock gene expression
in the liver under light-dark, whereas rhythmicity was restored under
disruptive light [198] (Figure 3).
These observations suggest that IF, similarly to CR, may affect the SCN clock.
This effect could possibly be mediated through a metabolic state generated by
the day of fast during IF regardless of the calories consumed, as discussed
earlier.

Altogether, the
findings in αMUPA mice suggest that reduced calories affect the SCN so it
becomes dominant over RF in the periphery only under disruptive light
conditions. In addition, IF affects peripheral rhythms depending on the timing
of food availability and light conditions, but regardless of the total daily
calorie consumption, suggesting that this regimen induces a metabolic state
that affects the SCN.

D. Temporal vs. quantitative food consumption and
circadian rhythms

Previous
publications have dealt with the issue of timed feeding and life span,
reporting that calorically restricted mice showed increased longevity whether
fed twice a day at daytime, once a day at daytime or nighttime, several times a
day at nighttime [231,232],
or three meals a week [233]. In
these studies, the low-calorie feeding was practically timed and confined to
the day or night, similarly to MUPA, or introduced in large intervals and
continued throughout life time allowing appropriate adaptation. As a result,
timed feeding was suggested to lead to high amplitude circadian rhythms and
increased life span [232,234].
However, others rejected any contribution of timed feeding to CR-induced
longevity [174,231].
The
uncoupling of timed meals from reduced calories could practically be achieved
only with animals, such as αMUPA, that spontaneously eat less. The results
obtained with αMUPA indicate that temporal and quantitative aspects of
food intake can be separately controlled. The timing of food intake is
controlled by the central biological clock, whereas a separate mechanism
appears to dictate the amount of food or calorie intake, that, in turn, could
entrain the SCN clock, as has experimentally been shown for calorically
restricted animals [191,192].
The
results achieved with IF suggest that IF can be beneficial when food is given
during the activity period of the animal, as explained above. Indeed, neuro-
and cardio-protection alongside increased fatty acid oxidation and improved
stress resistance have been induced after weeks of IF treatment when food was
introduced at the beginning of the activity period [205,235-237].
It
is noteworthy that cardio- and neuro-protection and life span extension
were also seen when food was introduced during the day, but after many months
of IF treatment [196,199],
so that the animals could adjust after such a prolonged treatment. In
light of these findings, we assume that the effect of IF on the SCN through a
metabolic change, as mentioned above, alongside the timed feeding might affect
the SCN to yield better-reset rhythms.

E.
Differences between αMUPA mice and calorically restricted rodents

Although
αMUPA mice exhibit reduced calorie intake and body fat, they show
remarkable differences in energy metabolism compared with CR-treated animals.
In particular, calorically restricted animals exhibit high levels of ghrelin [238,239],
but low levels of leptin [240,241]and insulin [173],
indicating an overall state of hunger. It is noteworthy that
leptin-deficient animals are long-lived under CR feeding regimen, suggesting
that leptin is not necessary for the CR-mediated benefits [242,243].
In addition, CR-treated mice exhibit high expression levels of PGC-1α and
no change in PPARγ levels in the liver [244,245].
All these findings are in sharp contrast with those found in αMUPA mice
that have low levels of ghrelin and high levels of leptin and insulin,
suggesting that αMUPA mice eat less because their metabolism is of
satiated rather than hungry animals (unpublished data). Nevertheless, one
aspect found to be common to both αMUPA mice and calorically restricted
animals are the low SIRT1 expression levels in the liver [246] and
high levels in the brain [247]. It
is noteworthy that the information regarding SIRT1 levels in the hypothalamus
of calorically restricted animals is still lacking, and data for peripheral
tissues is sometimes contradictory [124,246].
Results obtained with SIRT1-null micehave
recently suggested that this enzyme could be required for the in vivo
response to CR [248], and
transgenic mice over-expressing SIRT1 show a phenotype resembling calorically
restricted animals [249]. We
assume that, in calorically restricted mice, SIRT1 activity could be elevated
in the brain, possibly in the hypothalamus and SCN, through AMPK activation, as
AMPK can be activated in the hypothalamus under starvation conditions and high
ghrelin [112,121].
In αMUPA mice, that show low AMPK levels in the hypothalamus, the high
leptin levels could lead to SIRT1 elevation, as leptin is required for the
increase in SIRT1 protein levels in the hypothalamus under starvation [250].
Thus, although stimulated through different pathways in αMUPA and
calorically restricted mice, SIRT1 could act as a common factor modulating the
SCN clock and, as a result, longevity.

αMUPA
mice share also some similarities with those of Lou/C rats, both obesity-resistant long-lived rodents.

Figure
6. A
schematic model describing the effect of feeding regimens on longevity
through peripheral and SCN clock resetting. CR and IF reset
circadian rhythms in the periphery and the SCN. The synchronized, robust
circadian rhythms could be the mediator though which these feeding regimens
lead to aging attenuation and life span extension. RF resets circadian
rhythms only in the periphery, but its effect on life span is not known.

However,
there are some fundamental differences in Lou/C rats compared with αMUPA
mice, such as increased levels of PGC-1α and SIRT1 in the liver,
increased levels of ghrelin, and reduced levels of leptin and insulin in the
serum, although with some improved sensitivity for the latter two hormones [251,252].Overall, it seems that αMUPA mice are metabolically different from
Lou/C rats.

F. Role of
circadian rhythms in CR on health and longevity

The
capacity of CR to reset the SCN clock, as previously reported [191,192],and the pronounced circadian rhythms seen in the long-lived
αMUPA mice, pose the biological clock as a possible major factor
determining longevity of calorically restricted mice [194,195].
The beneficial effect induced by CR on health and longevity can be achieved by
appropriately resetting and synchronising a variety of hormonal, biochemical,
and physiological functions. In turn, some of these functions can feedback to
the biological clock in the periphery [143,153]
and the SCN [191,192]
and help sustain the rhythms. Indeed, the redox state affects the dimerization of
the two clock proteins CLOCK and BMAL1 in vitro [96]. As
SIRT1 has been linked to life span and suggested to mediate CR-induced effects [115,126,253],
it could be a candidate that modulates the clock of calorically restricted
animals, as discussed above. Clock resetting could
lead to robust circadian rhythms that are associated with young ages and
extended life span [15,16,19,195].

VI.
Conclusions

RF
entrains peripheral clocks due to temporal food consumption, whereas CR and IF
appear to synchronize the central pacemaker in the SCN, suggesting a role for a
metabolicstate imposed by low calories in
central clock entrainment. In αMUPA mice, reduced calories alone were
found not to be sufficient to sustain rhythms, unless feeding was spontaneously
timed at night, or timed at day through a restricted feeding protocol. Therefore,
it appears that when reduced calories are timed, as always occurs during CR and
IF regimens, clock adjustment can influence a wide variety of output systems,
so that cellular and physiological systems perform in a more synchronised and
appropriately reset manner. We assume that SIRT1 could be a key mediator in
clock synchronization at least under CR. Robust circadian rhythms can ensure a
better tissue and body homeostasis, and could constitute an important mediator
in aging attenuation and longevity extension (Figure 6).

CONFLICT OF INTERESTS STATEMENT

The authors of this
manuscript have no conflict of interest to declare.